Historically, the accepted method for determining the presence of microorganisms in a sample has been to streak the surface of nutrient agar or inoculate tubes of nutrient broth. After incubation at suitable temperatures, discrete areas of growth known as colonies appear on the agar surface or the tubes of broth become cloudy from the microorganism growth.
Although numerous chemical and physical microbial detection methods have been introduced including, carbon labelled glucose, ATP reaction, impedance measurements, electrochemical techniques, and pressure measurements, the agar plate and tubes of broth are still de rigueur in many laboratories. This is particularly true of clinical laboratories where blood samples are routinely introduced into containers of nutrient broth and growth visually monitored by the technician. Resistance to new techniques results partially from the biological fact that the number of organisms required to evoke a chemical or physical response is also essentially the same number present when turbidity becomes visually apparent in the broth. Thus, a test sample containing 10 to 100 cells/ml requires eight to ten hours incubation, or longer to reach 10.sup.5 to 10.sup.6 cells per ml, the point when visual turbidity occurs or a physical or chemical measurement is productive.
Another historical method that impacts the present invention is the growth of colonies on the horizontal surface of agar in a Petri dish. The solidifying agent, agar is generally used in concentrations of 1-2% and is referred to as firm or hard agar. However, little attention has been paid to the vertical subsurface growth of microorganisms in an agar medium. In studies on the effect of gravity on subsurface growth, Wilkins, J. R. et al ("Effect of Gravity on the Colonial Morphology of Staphylococci in Soft Agar" Applied Microbiology, Vol 18, No. 4; pages 680-681, October 1969) described the effect of agar concentration on the subsurface colony morphology of Staphylococcus aureus. As discussed therein, at an agar concentration of 0.54% compact spherical colonies were produced, but as the agar concentration was reduced, the morphology varied from a tear drop shape to elongated, diffuse colonies in soft agar at a concentration of 0.18% agar. In addition, even though colony morphology was shown to be influenced by agar concentration, the subsurface growth can also be used to determine the number of organisms in the test sample. The same guidelines that govern the accuracy of surface counts where colonies are determined to arise from single cells or clumps would also be applicable to subsurface colonies. The morphology of developing subsurface colonies aids in identification of the test organism in the sample.
Another prior art method of determining microorganism growth rate is the pour plate procedure. In this technique the sample is added to 20-30 ml of molten agar at 45.degree. C., mixed and at once poured into an empty Petri dish. After solidification at room temperature and incubation at 37.degree. C., the colonies are examined and counted. As the growth medium employed in the pour plate method contains 1.5% agar, the majority of subsurface colonies are lenticular in shape with a few compact or disk-shaped colonies. In the present invention the microbial detection process differs markedly from this classical pour plate technique in two important aspects. First, the concentration of agar has been reduced from the usual 1.5% to a range of 0.16 to 0.40% which is generally referred to as soft agar. Next, the vertical depth of the agar medium has been increased by an order of magnitude from 10 mm in a Petri dish to 100 mm in a specially designed culture cell. Thus, the combination of soft agar and increased vertical depth of the agar medium permits the development of subsurface colonies as described by Wilkins, et al (Applied Microbiology; vol. 18, supra). In this report at an agar concentration of 0.54% compact spherical colonies were produced and, as the agar concentration was reduced, the morphology varied from tear shape to elongated, diffuse colonies in soft agar at a concentration of 0.18% agar.
Systems are still needed to study the morphology of developing colonies as an aid in identification of test organisms in a test sample. Also, a system for detecting both aerobic and anaerobic organisms, where special media conditions are required to exclude oxygen, is needed in the art. Further, a need still exists for a simple method of automatically determining the types and numbers of various microbes by conceptually blending electronic video and computer technology with microbiology into a workable system.
Accordingly, it is an object of the present invention to provide an automated system for detecting and enumerating the number and types of viable microorganisms present in fluid clinical samples of blood, urine, body and spinal fluids, samples from polluted water, food processing plants and fermentation liquids.
Another object of the present invention is to provide a culture cell that permits subsurface growth of organisms in a vertical plane.
A further object of the present invention is to provide a culture cell having two chambers for separately and simultaneously growing aerobic and anaerobic organisms.
Another object of the present invention is to provide a dual compartment culture cell having a hinged closure that permits ready access to either chamber for adding growth media and removing microbial growth for further laboratory testing.
A further object of the present invention is the provision of a video camera for recording the subsurface growth in a culture cell.
An additional object of the present invention is the provision of a computer and video camera system for capturing, digitizing, and computer processing microbial growth information to determine the presence and quantity of a suspect microorganism from a test sample.